ES2912613T3 - Membrane support, kit for testing a liquid sample using the same, and method of manufacturing the same - Google Patents

Abstract

A membrane support (3) for detecting a target substance in a liquid sample, the membrane support (3) comprising: a flow path (2), a microstructure (7) formed at the bottom of the flow path ( 2), a detection zone (3y) for detecting the target substance in the liquid sample; and a marker provided in at least a part of the flow path (2) on an upstream side of the detection zone (3y), the marker being constituted of: a particle having a diameter of 500 nm or more and 100 µm or less, and an antibody or an antigen bound to the particle, wherein the antibody and the antigen are capable of specifically reacting with a target substance in the liquid sample, and a detection substance immobilized in the detection zone (3y) of the membrane support (3), such detection substance is capable of producing a color change by holding a target substance, such that the target substance to which the marker is to be attached can be detected in the area detection zone (3y) and the color of the detection zone (3y) will change when the marker is attached.

Description

DESCRIPTION

Membrane support, kit for testing a liquid sample using the same, and method of manufacturing the same Technical field

The present invention relates to a membrane support. Furthermore, a method for producing the membrane support, a test kit comprising the membrane support, and the method for producing the test kit are described.

prior art

Recently, Point-of-Care Testing (POCT) reagents using, for example, antigen-antibody reactions to determine contraction of infectious diseases, pregnancy, blood sugar level, and the like have drawn attention. POCT reagents, which are test reagents used near subjects or used directly by subjects, have features such as ability to determine test results in a short time, simple operation, and low cost. By virtue of these characteristics, POCT reagents are frequently used, for example, in medical examinations at the stage of mild symptoms and regular medical examinations, and are used as an important examination tool in home health care which is expected to expand to from now on.

In most POCT reagents, the determination is made by introducing a liquid sample, such as blood, into a test kit and detecting a predetermined target substance contained in the liquid sample. As a method for detecting a predetermined target substance from a liquid sample, immunochromatography is frequently used. Immunochromatography is a technique for detecting a target substance by adding a liquid droplet to a membrane support of a test kit, allowing the liquid droplet to move on the membrane support, allowing a target substance to bind to marker particles (hereinafter also referred to simply as the particle) attached to an antibody or antigen that specifically reacts with the target substance and suspended or dissolved in a liquid sample, and allowing these particles to bind more specifically to a substance (hereinafter referred to as detection substance) immobilized on the membrane support to cause a change in color or weight, and detect the change. The detection substance may also be called a reagent.

As a technique for detecting a target substance, a technique for detecting a color change produced using label particles is well known. Examples of marker particles include colored latex particles, fluorescent latex particles, and metallic colloidal particles.

In the detection method using marker particles as mentioned above, it is known that as the size of the marker particles increases, the sensitivity improves. Patent Literature 1 to 2 show that, in immunodiagnosis using turbidimetry, if the diameter of the latex increases, the intensity of light scattering increases and the sensitivity increases.

As a POCT reagent for optically determining a color change, a lateral flow type kit using a nitrocellulose membrane is often used. The nitrocellulose membrane has many micropores and a liquid sample moves through the micropores with the help of capillary force. In contrast, the pores of the nitrocellulose membrane are as fine as on the order of several pm. Because of this, there was an upper limit to the diameter of the marker particles.

Furthermore, the nitrocellulose membrane, which is derived from a natural product, has pores of non-uniform size. Due to this, the diameter of the marker particles to be developed was set to be smaller so as not to cause problems such as obstructions; while the sensitivity was low.

Patent literature 1 to 4 shows that the measurable range of concentration in turbidimetry can be extended by using two or more types of latex particles of different diameter in combination.

However, in immunochromatography, since a nitrocellulose membrane support having micropores was used, the size of a label particle is limited. The effect of the combined use of two or more types of latex particles of different diameter on performance was not yet known.

Patent Literature 5 discloses a liquid sample test kit having a microstructure formed therein instead of using a nitrocellulose membrane support; however, the effect of latex diameter on performance is not disclosed.

The 6 and 7 patent literature discloses membrane supports with immobilized particles.

Patent literature 8 discloses another membrane support.

Patent literature 9 discloses a membrane support with colored latex particles.

citation list

Patent bibliography

Patent Literature 1: Japanese Examined Patent Publication No. S63-14783

Patent Bibliography 2: Japanese Patent No. 2588l 74

Patent Literature 3: Japanese Patent No. 3513075

Patent Literature 4: Japanese Patent No. JP3652029B

Patent Literature 5: WO 2016/098740

Patent Literature 6: US 7824611 B2

Patent Literature 7: US 2005/136552 A1

Patent Literature 8: WO 2011/045436 A1

Patent Literature 9: WO9119980

Summary of the invention

technical problem

The present invention was made in view of the above mentioned problems and is directed to providing a membrane support which allows highly sensitive detection.

Solution to the problem

More specifically, the present invention is as follows.

A membrane support for a test kit for detecting a target substance in a liquid sample, the membrane support comprising:

a flow path,

a microstructure formed at the bottom of the flow path,

a detection zone for detecting the target substance in the liquid sample; Y

a marker provided in at least a part of the flow path on an upstream side of the detection zone, the marker consisting of:

a particle having a diameter of 500 nm or more and 100 pm or less, and an antibody or an antigen bound to the particle, wherein the antibody and the antigen can specifically react with a target substance in the liquid sample, wherein

a detection substance is immobilized on the detection zone of the membrane support, which detection substance can cause a color change by retaining a target substance, such that the target substance to which the label is to bind can be detected in the detection zone and the color of the detection zone will change after joining the marker.

According to a preferred embodiment, the membrane support is disclosed in which the average horizontal distance between adjacent convex parts constituting the microstructure is at least 3 times the particle diameter and 300 pm or less.

According to another preferred embodiment, the membrane support is disclosed wherein the particle is one or more selected from the group consisting of a colored latex particle and a fluorescent latex particle.

According to another preferred embodiment, the membrane support is disclosed in which the detection zone can produce a color change when the target substance is detected, in such a way that the target substance to which the marker is going to bind can be detected. in the detection zone and where the color of the detection zone will change after joining the marker.

According to a further aspect, a method of making a membrane support is disclosed which comprises immobilizing a detection substance capable of producing a color change by holding a target substance in the detection zone of the membrane support as described above.

Furthermore, a test kit comprising the membrane support and a method for producing the membrane support are described.

Advantageous effects of the invention

According to the membrane support of the present invention, a highly sensitive test can be carried out.

Brief description of the drawings

Figure 1 shows a schematic top view of a test kit comprising the membrane support according to an embodiment of the present invention.

Figure 2 shows a schematic top view of a membrane support that is an embodiment of the present invention.

Figure 3 shows (a) a plan view (top view) of microstructures which is an embodiment of the present invention; and (b) a perspective view of a convex portion constituting the microstructure shown in (a).

Figure 4 shows (a) a plan view (top view) of a microstructure which is an embodiment of the present invention; and (b) a perspective view of a convex portion constituting the microstructure shown in (a).

Figure 5 shows (a) a plan view (top view) of a microstructure which is an embodiment of the present invention; and (b) a perspective view of a convex portion constituting the microstructure shown in (a).

Figure 6 shows (a) a plan view (top view) of a microstructure which is an embodiment of the present invention; and (b) a perspective view of a convex portion constituting the microstructure shown in (a).

Figure 7 shows a sectional view of a membrane support having a microstructure which is an embodiment of the present invention.

Figure 8 shows (a) a top view of microstructures according to one embodiment of the present invention; and shows (b) a sectional view of a membrane support having the microstructures shown in (a). Description of achievements

Embodiments of the present invention will be described below.

A membrane support according to one embodiment is a membrane support for a liquid sample test kit, which detects a target substance in a liquid sample.

Herein, the target substance, which is not limited, may be any substance as long as it can undergo antigen-antibody reaction with various pathogens, various clinical markers, antibodies or antigens. Examples of the target substance include, but are not particularly limited to, antigens of viruses such as influenza virus, norovirus, adenovirus, RS virus, HAV, HBs, and HIV; antigens from bacteria such as MRSA, group A streptococci, group B streptococci, and Legionella bacteria; toxins produced by bacteria, Mycoplasma, Chlamydia trachomatis, hormones such as human chorionic gonadotropin; and C-reactive protein, myoglobin, myocardial troponin, various tumor markers, agrochemicals, environmental hormones, Treponema pallidum (TP) antibodies (TPAb), and anti-pylori antibodies. If the target substance is particularly an item that needs to be detected and treated quickly, such as influenza virus, norovirus, C-reactive protein, myoglobin and myocardial troponin, the liquid sample test kit and membrane support of according to the realization are extremely useful. The target substance can be an antigen, which only induces an immune response, or it can be a hapten, which cannot induce an immune response by itself but can induce an immune response if bound to an antibody through an antigen-antibody reaction. . The target substance is usually suspended or dissolved in a liquid sample. The liquid sample may be a sample obtained by suspending or dissolving the target substance in, for example, a buffer solution.

The liquid sample test kit according to the embodiment (hereinafter also simply referred to as "test kit") detects a target substance in a liquid sample. Figure 1 is a schematic top view of a test kit. For example, as shown in Figure 1, a test kit 18 comprises a membrane holder 3 and a box 18a to accommodate the membrane holder 3. The membrane holder 3 has, on its surface, a landing zone 3x wherein a drop of a liquid sample and a detection zone 3y for detecting a target substance in a liquid sample are supplied. The placement zone 3x is exposed in a first opening 18b of the box 18a. The detection zone 3y is exposed in the second opening 18c of the case 18a.

Figure 2 is a schematic top view of the membrane support 3. As shown in Figure 2, the membrane support 3 has at least one flow path 2 for transporting a liquid sample and a marker (not shown, details will be described later) provided on the membrane support to successfully react with a target substance. The label consists of the particle and an antibody or antigen bound to the particle. The antibody and the antigen may be an antibody and an antigen, each of which react specifically with a target substance in a liquid sample. The marker may be provided in at least a part in the flow path 2 of the membrane support 3. In the lower part of the flow path 2, a microstructure (not shown, details will be described later) is provided. The microstructure is present at least between the placement zone 3x and the detection zone 3y. The microstructure can be provided over the entire surface of the membrane support 3. The entire surface of the membrane support 3 can serve as a flow path 2 for a liquid sample. Due to the microstructure, capillary action occurs. A liquid sample is transported from the 3x placement zone to the 3y detection zone (along the transport direction d) through the microstructure with the help of capillary action produced by the microstructure. During the transport process, a target substance in a liquid sample binds to a marker. When the target substance to which the label binds is detected in detection zone 3y, the color of detection zone 3y changes.

The whole shape of the membrane support 3 is not particularly limited; however, the shape can be, for example, a polygon such as a rectangle, circle, or ellipsoid. If the membrane support 3 is a rectangle, the length (length of the shortest side) L1 of the membrane support 3 may be, for example, 2 mm or more and 100 mm or less, and the width (length of the shortest side long) L2 of the membrane support 3 may be, for example, 2 mm or more and 100 mm or less. The thickness of the membrane support, excluding the heights of the microstructure, may be, for example, 0.1 mm or more and 10 mm or less.

Figs. 3 to 6 and 8 each show a microstructure provided at the bottom of the flow path according to the embodiment and an example of convex portions constituting the microstructure. In each of Figures 3 to 6, (a) is a plan view (top view) of the microstructure; and (b) is a perspective view of one of the convex parts that constitute the microstructure. As shown in Figures 3 to 6 and 8, a microstructure 7 is an assembly of convex parts 8. More specifically, the membrane support 3 has a flat part 9 corresponding to the lower part of the flow path 2 of a sample. liquid and a plurality of convex parts 8 corresponding to the flat part 9 The space between the convex parts 8 serves as a flow path 2 for transporting a liquid sample along the surface of the membrane support 3 with the help of capillary action . In other words, the space in the microstructure 7 serves as a flow path 2 to transport a liquid sample along the surface of the membrane support 3 by capillary action. The convex portions 8 can be arranged on the surface of the membrane support 3 in a regular or translationally symmetric manner.

The shape of the convex parts 8 constituting the microstructure 7 can be freely selected. Examples of the shape of the convex portions 8 include a cone, a polygonal pyramid, a truncated cone, a truncated polygonal pyramid, a cylinder, a polygonal column, a hemisphere, and a semiellipsoid. For example, the shape of the convex portions 8a may be a cone as shown in Fig. 3. For example, the shape of the convex portions 8b may be a square pyramid as shown in Fig. 4. For example, the shape of the convex portions 8c may be a hexagonal pyramid as shown in Fig. 5. For example, the shape of the convex portions 8d may be a horizontally long triangular prism (triangular prism placed in such a way that one side surface of the triangular prism ( a rectangular surface) is in contact with the flat part 9) as shown in Figure 6. For example, the shape of the convex parts 8e can be a cylinder, as shown in Figure 8. Because when the microstructure 7 is looking down (view from above), the entire surface of the membrane support 3 can be seen, and a color change when a target substance is detected can be easily verified by optical means, a cone structure like a cone and a polygonal pyramid is suitable as the shape of the convex portions 8, among the shapes mentioned above.

The shape of the convex portions 8 constituting the microstructure 7 need not be a geometrically precise shape, and may be a shape having a rounded corner and a shape having microconvex-concaves on the surface.

The diameter 4 of each of the lower surfaces 10 of the convex portions 8 constituting the microstructure 7 may be 10 pm or more and 1000 pm or less, and more preferably 15 pm or more and 1000 pm or less. The diameter 4 of the lower surface 10 of the convex part 8 may vary (be different from each other) among a plurality of convex parts 8 within the above range. If the diameter 4 of each of the bottom surfaces 10 of the convex portions 8 is 10 pm or more, the microfabrication cost of a mold for forming the microstructure 7 decreases and an infinite number of microstructures 7 can be easily and uniformly formed in the surface. of the large area membrane support 3. Therefore, a microstructure consisting of convex portions 8 having the bottom surface 10 of 10 pm or more in diameter 4 is more practical. If the diameter of each of the bottom surfaces 10 of the convex portions 8 is 10 pm or Furthermore, the capillary force required to move a liquid sample tends to increase. If the diameter 4 of each of the lower surfaces 10 of the convex portions 8 is 1000 pm or less, the volume of metal discarded from a metal member at the time of forming a mold can be reduced, with the result that the manufacturing costs for the mold and the membrane support 3 can be eliminated. If the diameter of each of the bottom surfaces 10 of the convex portions 8 is 1000 pm or less, the area of the flow path 2 in the membrane support 3 can be reduced, with the result that a kit of liquid sample test 18. This is advantageous to send own liquid sample test kit 18.

The diameter 4 of each of the lower surfaces 10 of the convex portions 8 is defined as the representative length of the lower surface 10 of the convex portion 8. The representative length defining the lower surface 10 is a diameter if the shape of the lower surface 10 is a circle; the length of the shorter side if the shape is a triangle or a rectangle; the length of the longest diagonal line if the shape is a polygon of a pentagon or more; and a maximum length of the bottom surface 10 in the case of shapes other than those mentioned above.

Figure 7 is an aligned sectional view of the membrane support having a microstructure 7a taken along the line VII-VII shown in Figure 3. As shown in Figure 3 and Figure 7, if the shape of the convex part 8a is a cone, the diameter 4a of the lower surface 10a of the convex part 8a corresponds to the diameter of the bottom (circle) of the cone. As shown in Figure 4, if the shape of the convex part 8b is a regular square pyramid, the diameter 4b of the bottom surface 10b of the convex part 8b is the length of the sides of the bottom surface (regular square) 10b. . As shown in Fig. 5, if the shape of the convex part 8c is a regular hexagonal pyramid, the diameter 4c of the bottom surface 10c of the convex part 8c is the length of a diagonal line (length of the longest diagonal line ) passing through the center of the bottom surface (regular hexagon) 10c. As shown in Figure 6, if the shape of the convex part 8d is a horizontally long triangular prism, the diameter 4d of the bottom surface 10d of the convex part 8d is the length of the shortest side of the bottom surface (rectangle) 10d (in Figure 6, the length of the side perpendicular to the transport direction d of a liquid sample). Figure 8(b) is an aligned sectional view of the membrane support having a microstructure 7e taken along line A-A shown in Figure 8(a). As shown in Fig. 8, if the shape of the convex portions 8e is a cylinder, the diameter 4e of the bottom of the convex portion 8e is the diameter of the circular bottom of the cylinder.

The height 6 of each of the convex portions 8 constituting the microstructure 7 is preferably 10 pm or more and 500 pm or less, and more preferably 15 pm or more and 500 pm. The height 6 of the convex portions 8 may vary (be different from each other) among a plurality of convex portions 8 within the above range. If the height 6 of the convex portions 8 is 10 pm or more, the volume of the flow path 2 increases, with the result that a liquid sample can be developed in a shorter time. If the height 6 of each of the convex portions 8 is 500 pm or less, the time and cost for forming the microstructure 7 can be reduced, with the result that it is easy to prepare the microstructure 7.

The height 6 of the convex part 8 is defined as a maximum length of the convex part 8 in the direction perpendicular to the flat part 9. As shown in Figure 3 and Figure 7, if the shape of the convex part 8a is a cone, the height 6a of the convex part 8a is a maximum length of the convex part 8a in the direction perpendicular to the flat part 9 (the height of the cone). As shown in Fig. 4, if the shape of the convex part 8b is a square pyramid, the height 6b of the convex part 8b is a maximum length of the convex part 8b in the direction perpendicular to the flat part 9 (the height of the square pyramid). As shown in Figure 5, if the shape of the convex part 8c is a hexagonal pyramid, the height 6c of the convex part 8c is a maximum length of the convex part 8c in the direction perpendicular to the flat part 9 (the height of the hexagonal pyramid). As shown in Figure 6, if the shape of the convex part 8d is a horizontally long triangular prism, the height 6d of the convex part 8d is a maximum length of the convex part 8d in the direction perpendicular to the flat part 9 ( the height of the horizontally long triangular prism). As shown in Fig. 8, if the convex part 8e is a cylinder, the height 6e of the convex part 8e is the maximum length of the convex part 8e in the direction perpendicular to the flat part 9 (the height of the cylinder).

The average horizontal distance between adjacent microstructures, in the case where the horizontal distance between adjacent microstructures (for example, between convex parts 8) varies depending on the height of the convex parts 8, such as a cone, a hemisphere and a semi-ellipsoid, is specified as a mean value of the farthest horizontal distance 5A between adjacent microstructures (nearest center-to-center distance) and the nearest horizontal distance 5B between adjacent microstructures, i.e. (5A+5B)/2, as shown in Figures 3 to 7.

The mean horizontal distance between adjacent microstructures, in the case where the horizontal distance between adjacent microstructures does not vary depending on the height of the microstructure, such as a column, is specified as the distance of the 5C space between microstructures (convex parts 8) as it is shown at the distance 5C of the space between adjacent microstructures in Figure 8 (the convex portions 8 are cylinders).

The average horizontal distance between adjacent microstructures can be at least 3 times or at least 4 times and at most 600 times or at most 500 times the diameter of the particle. If the mean horizontal distance between adjacent microstructures is at least 3 times the particle diameter, the risk that the flow of the liquid sample is interrupted by the resistance of the particle is further suppressed, with the result that the support membrane cannot be used as a kit.

The average horizontal distance between adjacent microstructures may be 1.5 pm or more, 2.0 pm or more, or 2.5 pm or more, and may be 300 pm or less, 250 pm or less, or 200 pm or less. If the mean horizontal distance between adjacent microstructures is 300 pm or less, the risk of the area in which a liquid sample is in contact with the flow path is suppressed and a reduction in capillary force is suppressed, with the result that the liquid sample cannot move.

The average horizontal distance between adjacent microstructures is preferably at least 3 times the particle diameter and 300 pm or less, and more preferably at least 4 times the particle diameter and 300 pm or less.

The microstructure 7 and the membrane support 3 of the liquid sample test kit 18 of the embodiment may be made of a thermoplastic. In other words, the membrane support 3 having the microstructure 7 can be produced by processing a film-like base material made of a thermoplastic. Examples of the processing method include thermal printing, UV printing, injection molding, etching, photolithography, machine cutting, and laser processing. Of these, thermal printing to a thermoplastic is suitable as a method for applying precise processing at low cost. Examples of the thermoplastic include polyester resin, polyolefin resin, polystyrene resin, polycarbonate resin, fluororesin and acrylic resin. More specifically, various types of resins can be used, including polyethylene terephthalate (PET), a cycloolefin polymer (COP), polypropylene (PP), polystyrene (PS), polycarbonate (PC), polyvinylidene fluoride (PVDF), and polymethyl methacrylate. (PMMA).

In the case of processing using a mold, such as impression molding or injection molding, since the top of a cone is narrow compared to the bottom, the volume of metal scrapped in forming the mold is smaller than a columnar mold which has the same bottom area, and therefore the mold can be made cheaply with a cone. In this case, a target substance can be detected in a liquid sample at low cost.

The label consists of the particle and an antibody or antigen bound to the particle. The label can bind to a target substance through the antibody or antigen.

The marker may be provided to at least a portion of the flow path on the upstream side of detection zone 3y (between drop zone 3x and detection zone 3y (including drop zone 3x)). The marker may be provided to at least a portion of the 3x landing zone or over the entire 3x landing zone. Alternatively, the label may be provided together with a member for use in the test kit 18 to flow path 2. The label that reacted with (bound to) a target substance is retained by a detection substance (via the reaction (binding) of the detection substance with the target substance) in the detection zone 3y. In this way, a color change (color produced by a marker) is produced in the detection zone 3y.

The antibody or antigen may be a binding fragment. The binding moiety refers to a moiety that can specifically bind to a target substance; for example, reference is made to an antigen-binding fragment of an antibody or an antibody-binding fragment of an antigen.

Examples of the particle include colloidal particles and latex particles. The particles may have magnetic properties or fluorogenicity. Examples of colloidal particles include metallic colloidal particles such as colloidal gold particles and colloidal platinum particles. The particles are preferably latex particles in view of particle size control, dispersion stability and binding ability. The material for latex particles is not particularly limited; however, polystyrene is preferred.

In view of visibility, the particles are preferably colored particles or fluorescent particles, and more preferably colored particles. Colored particles are satisfactory if the color thereof is detectable with the naked eye. Fluorescent particles are satisfactory if they contain a fluorescent substance. The particles can be colored latex particles or fluorescent latex particles. If the particles are colored latex particles, the aforementioned color change is adequately detected visually. If the particles are fluorescent latex particles, the color change mentioned above is suitably detected by measuring the intensity of fluorescence.

The particle diameter is 500 nm or more and 100 pm or less. The particle diameter may be 600nm or more, 800nm or more, 1pm or more, 1.2pm or more, 1.5pm or more, 2pm or more, or 2.5pm or more and 80 pm or less, 60 pm or less, 50 pm or less, 20 pm or less, 10 pm or less, or 5 pm or less. The particle diameter is preferably 600 nm or more and 80 pm or less, 800 nm or more and 60 pm or less, 1 pm or more and 50 pm or less, 1.2 pm or more and 20 pm or less or 2 pm or more and 10 pm or less. If it is less than 500 nm, the color change produced when the particle (according to claim 1) is immobilized in the detection zone 3y is small and the detection sensitivity may be reduced. If the diameter is greater than 100 pm, the flow of liquid sample is interrupted by the resistance of the particles, with the result that the membrane support does not can be used as a kit.

Particle diameter refers to the diameter of a particle measured by dynamic light scattering. Dynamic light scattering is described, for example, in Japanese Patent No. 5147011 and Beckman Coulter, Inc. home page ("measurement principle of dynamic light scattering", <URL: https://beckman.jp /column/particle/m_principle/>).

To perform a highly sensitive test, it is preferable to use particles of different diameters in combination. According to the present inventors, the reason is presumably as follows. Since the contact area between a detection substance and a microstructure varies depending on the particle diameter, the time required for an antigen-antibody reaction varies. The reaction time is affected by the flow rate of a liquid sample, and the flow rate changes depending on the height in the flow path from the bottom. Since the rate at which a reaction with a detection substance easily proceeds varies depending on the diameter of the particle, the height at which adhesion to a microstructure can be easily performed varies. As a result, if a plurality of types of particles are developed in a microstructure, the particles are separated into those that are easily adhered to the upper part of the structure and those that are easily adhered to the lower part of the structure. Due to this, the total adhesion area is increased compared to the case where only one type of particle is developed. As a result, a target substance can be easily detected and the sensitivity is improved.

It is preferable that a plurality of types of particles of different diameter are used; and more preferable that two types of particles different in particle diameter are used. In the case that two types of particles with different particle diameters are used, as long as the particles having a smaller diameter are represented by PI and the particles having a larger diameter are represented by P2, the mass ratio (P1 /P2) is preferably from 10/90 to 90/10, more preferably from 30/70 to 70/30 and even more preferably 50/50.

As described above, the membrane support 3 has microstructures 7 formed on one of the surfaces of the membrane support 3, the flow path 2 formed by the microstructures 7 and used to transport a liquid sample and marker provided on the support membrane 3 to successfully react with a target substance and have the particle and an antibody or antigen bound to the particle. The particle diameter is 500 nm or more and 100 gm or less. The membrane support 3 may be a membrane support 3 for the liquid sample test kit 18 for detecting a target substance in a liquid sample.

In the liquid sample test kit 18 according to the embodiment, the detection zone 3y of the membrane support 3 produces a color change when a target substance is detected. The color change may be a color change that can be observed by optical means.

As optical means, two methods are mainly mentioned: a visual determination means and a fluorescence intensity measurement means. In the case of visual determination, it is preferable to produce a color change expressed by a color difference (AE described in JIS Z8781-4:2013) of 0.5 or more between two color stimuli before and after detection when color is measured by the CIE1976L*a*b*color space color system. If the color difference is 0.5 or more, the visual determination of the color difference can be done easily. In the case of a determination based on fluorescence intensity measurement, it is preferable to produce a color difference that satisfies a relationship between the fluorescence intensity (Fl1) in the detection zone 3y and the fluorescence intensity ( Fl2) in the upstream region and the downstream region adjacent to detection zone 3y, (Fl1/Fl2) = 10/1 or more. If the ratio is 10/1 or more, the signal and noise can be easily separated.

To prepare the detection zone 3y on the membrane support 3 of the liquid sample test kit 18 of the embodiment, a detection substance is immobilized in at least part of the flow path 2, in one embodiment. More specifically, a detection substance that detects a target substance is immobilized in the detection zone 3y. A color change occurs in the detection zone 3y by keeping a target substance by the detection substance (by reacting with the detection substance) in the detection zone 3y.

In other words, a method for producing a membrane support 3 for a liquid sample test kit 18 comprises a step of immobilizing, in the detection zone 3y, a detection substance that produces a color change by keeping the target substance in the detection zone 3y. Since a detection substance (reagent) according to claim 1 is efficiently immobilized in the detection zone 3y, the surface treatment can be previously applied to the site of the membrane support 3, where the detection zone is to be provided. 3y.

The surface treatment method is not limited, and for example, various methods such as UV irradiation, UV/ozone treatment, various plasma treatments and surface modification can be used. with, for example, 3-aminopropyltriethoxysilane or glutaraldehyde.

In the embodiment, as the detection substance (reagent), for example, an antibody is mentioned. The antibody is an antibody that binds to a target substance through an antigen-antibody reaction, and may be a polyclonal antibody or a monoclonal antibody.

The color change in the detection zone 3y can be caused by a marker having a particle and an antibody or an antigen bound to the particle. The color change occurs, for example, by keeping a label by a detection substance (through a reaction with (binding to) the detection substance) in the detection zone 3y and producing a color.

One method of testing a liquid sample according to one aspect of the embodiment is a test method using test kit 18.

The method for testing a liquid sample using the test kit 18 may comprise a step of preparing a mixed liquid sample by mixing the liquid sample and a label that specifically binds to a target substance in the liquid sample to mutually bind the target substance and marker; a step of supplying a drop of the mixed liquid sample to the placement zone 3x provided on the membrane support 3; a step of transporting the mixed liquid sample from the placement zone 3x to the detection zone 3y through the microstructure 7; and a step of detecting a color change (marker color) in the detection zone 3y.

Alternatively, the above test method may comprise a step of supplying a drop of a liquid sample to the 3x placement zone on the surface of the membrane support 3; a step of transporting the liquid sample from the placement zone 3x to the detection zone 3y through the microstructure 7 with the help of capillary action exerted by the microstructure 7 (convex portions 8) formed on the surface of the membrane support 3; and a step of binding a target substance in a liquid sample to the label via the antibody or the antigen, further binding the target substance to an immobilized reagent in the detection zone 3y and detecting a color change in the detection zone 3y (optically determining the presence or absence of color change).

In the mutual binding step of a target substance and a marker in the above test method, a method of mixing a liquid sample and the marker is not particularly limited. For example, a method of adding a liquid sample into a container containing the tracer or a method of mixing a liquid containing, for example, a tracer and a liquid sample may be employed. Alternatively, a filter is inserted into a drip opening of a container containing, for example, a liquid sample, and a label may be immobilized on the filter.

examples

Embodiments will be described; however, the embodiments are not limited by these experimental examples.

[Experiment example 1]

<Preparation of the mold>

The mold was prepared by laser processing and machine cutting. The mold is made of A5052 aluminum alloy. In the center of the mold, concave conical parts having a diameter of 25 pm and a depth of 30 pm are provided to the nearest center-to-center distance (the farthest horizontal distance) of 30 pm, the nearest horizontal distance ( the nearest horizontal distance) between adjacent microstructures of 5 pm and a mean distance of 17.5 pm, in a staggered arrangement, within the interval of 3 cm x 3 cm, as shown in Figure 3.

In order to easily separate the mold and a thermoplastic without failure at the time of transfer printing, a release treatment was applied to the convex-concave surface of the mold. The release treatment was carried out by soaking the mold in Optool HD-2100TH manufactured by Daikin Industries Ltd. for about one minute, drying it, and then allowing the mold to stand overnight.

<Microstructure transfer printing>

Using the mold obtained as mentioned above, the microstructure was transfer printed onto a thermoplastic. As the thermoplastic, polystyrene (Denka styrene sheet manufactured by Denka Company Limited, film thickness 300 pm) was used. Thermal printing was used as processing method. As the apparatus, the X-300 manufactured by SCIVAX was used. Transfer printing was carried out at a molding temperature of 120°C and an applied pressure of 5.5 MPa for 10 minutes. After transfer printing, the thermoplastic and mold were cooled to 80°C while pressure was applied, and then the pressure was removed to prepare a membrane support.

In the fabricated membrane support, the farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are shown in Table 1. The convex parts are cones. The thickness of the membrane support except the height of the microstructure (convex parts) is 0.2 mm.

<Preparation of detection area>

On the positions of the previously manufactured membrane support at a distance of 0.6 cm and 1.0 cm from the lower edge, a suspension solution of antibodies against NPs of influenza type A and a suspension solution of antibodies against NPs of type A were applied. influenza type B in a width of 3 cm (coating amounts were each 3 pl) and sufficiently dried with hot air to immobilize the detection substances.

<Marker Preparation>

Purified anti-influenza A virus NP antibodies (other antibody as used above) and purified anti-influenza B virus NP antibodies (other antibody as used above) were used. Anti-influenza A virus NP antibody was covalently labeled with red latex particles (SC-042-R polystyrene latex particles, colored latex particles, manufactured by JSR Life Sciences Corporation) with a particle diameter of 0 0.2 pm, suspended in a Tris buffer solution containing a sugar, a surfactant and a protein in such a way that the concentration of the latex particles became 0.025% by mass/v, and ultrasonically treated to prepare a sufficiently dispersed and suspended anti-type A marker. The anti-type B marker was prepared in a similar manner by labeling an anti-influenza type B NP antibody with blue latex particles.

The anti-type A marker and the anti-type B marker were mixed and applied to glass fiber having a size of 3 cm x 1 cm (33GLASS No. 10539766, manufactured by Schleicher & Schuell) in an amount of 50 pl per square centimeter and dried well with hot air to produce a marker pad. Subsequently, the marker pad was overlaid with the edge part (only 2 mm) of the prepared membrane support and cut into strips having a width of 5 mm with a cutter to prepare integrated liquid sample test kits.

<Detection evaluation>

On the edge of the liquid sample test kit prepared as mentioned above, the liquid sample (100 pl) was dropped. The liquid sample was diluted with a specimen suspension solution attached to Quick navi-Flu manufactured by Denka Seiken Co., Ltd., as a dilution solution. A maximum dilution rate (maximum permissible dilution rate of visible determination of type A) was obtained, at which the presence or absence of a colored line cannot be visually observed 10 minutes after the start of the test, increasing the rate Influenza A/Beijing/32/92 (H3N2) virus test dilution ratio of 2 x 104. A test was performed at a dilution rate of 1/2 as low as the maximum dilution rate for obtain the time until the concentration of the colored line stabilizes (time until the concentration of type A stabilizes) from the start of the test, as the detection time. The results are shown in Tables 1 and 2.

To one edge of the liquid sample test kit made as described above, a liquid sample (100 pl) was added dropwise. The liquid sample was diluted with a specimen suspension solution attached to Quick navi-Flu manufactured by Denka Seiken Co., Ltd., as a dilution solution. A maximum dilution rate (maximum permissible dilution rate of visible determination of type B) was obtained, at which the presence or absence of a colored line cannot be visually observed 10 minutes after the start of the test, increasing the dilution rate of influenza B virus type B/Shangdong/7/97 of 2 x 103. A test was carried out at a dilution rate of 1/2 as low as the maximum dilution rate to obtain the time until it stabilizes. the concentration of the colored line (time until the concentration of type B stabilizes) from the start of the test, as the detection time. The results are shown in Table 1.

The detection time in Table 1 refers to a mean value of the time until the concentration of type A stabilizes and the time until the concentration of type B stabilizes.

General evaluations were carried out based on the permissible dilution rates of visible determination according to the following criteria and the results are shown in Table 1.

A: The visible determination allowable dilution rate is 7 x 103 or more, and the detection time is 7 minutes or less.

B: None of A, C and D apply

C: The visible determination allowable dilution rate is more than 3 x 103 and 4 x 103 or less, or the detection time is 10 minutes or more.

D: The allowable dilution rate of visible determination is 3 x 103 or less.

[Experiment example 2]

The experiment was carried out under the same conditions as in Experimental Example 1 except that the particle diameter of the latex used in the microstructure of Experimental Example 1 was specified as 200 pm. In the fabricated membrane support, the convex portions were cone-shaped. The farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are shown in the Table.

[Experiment example 3]

The experiment was carried out under the same conditions as in Experimental Example 1 except that the particle diameter of the latex used in the microstructure of Experimental Example 1 was specified as 0.5 pm. In the fabricated membrane support, the convex portions were cone-shaped. The farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are shown in the Table.

[Experimental Example 4]

The experiment was carried out under the same conditions as in Experimental Example 1 except that the particle diameter of the latex used in the microstructure of Experimental Example 1 was specified as 1 pm. In the fabricated membrane support, the convex portions were cone-shaped. The farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are shown in the Table.

[Experimental Example 5]

The experiment was carried out under the same conditions as in Experimental Example 1 except that the particle diameter of the latex used in the microstructure of Experimental Example 1 was specified as 5 pm. In the fabricated membrane support, the convex portions were cone-shaped. The farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are shown in the Table.

[Experimental Example 6]

The experiment was carried out under the same conditions as in Experimental Example 1, except that the microstructures of Experimental Example 1 were specified as concave-conical with a diameter of 80 pm, a farthest horizontal distance of 100 pm, a farthest horizontal distance close distance of 20 pm, a mean distance of 60 pm, and a depth of 100 pm, and that the diameter of the latex particles was specified as 1 pm. In the fabricated membrane support, the convex portions were cone-shaped. The farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are shown in the Table.

[Experiment example 7]

The experiment was carried out under the same conditions as in Experimental Example 1, except that the microstructures of Experimental Example 1 were specified as concave-conical with a diameter of 80 pm, a farthest horizontal distance of 100 pm, a farthest horizontal distance close distance of 20 pm, a mean distance of 60 pm and a depth of 100 pm and that the diameter of the latex particles was specified as 5 pm. In the fabricated membrane support, the convex portions were cone-shaped. The farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are shown in the Table.

[Experiment example 8]

The experiment was carried out under the same conditions as in Experimental Example 1, except that the microstructures of Experimental Example 1 were specified as concave-conical with a diameter of 80 pm, a farthest horizontal distance of 100 pm, a farthest horizontal distance close distance of 20 pm, a mean distance of 60 pm and a depth of 100 pm, and that the diameter of the latex particles was specified as 20 pm. In the fabricated membrane support, the convex portions were cone-shaped. The farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are show in the table.

[Experiment example 9]

The experiment was carried out under the same conditions as in Experimental Example 1, except that the microstructures of Experimental Example 1 were specified as concave-conical with a diameter of 300 pm, a farthest horizontal distance of 450 pm, a furthest horizontal distance close distance of 150 pm, a mean distance of 300 pm and a depth of 450 pm, and that the diameter of the latex particles was specified as 5 pm. In the fabricated membrane support, the convex portions were cone-shaped. The farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are shown in the Table.

[Experimental Example 10]

The experiment was carried out under the same conditions as in Experimental Example 1, except that the microstructures of Experimental Example 1 were specified as concave-conical with a diameter of 300 pm, a farthest horizontal distance of 450 pm, a furthest horizontal distance close distance of 150 pm, a mean distance of 300 pm and a depth of 450 pm, and that the diameter of the latex particles was specified as 20 pm. In the fabricated membrane support, the convex portions were cone-shaped. The farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are shown in the Table.

[Experiment example 11]

The experiment was carried out under the same conditions as in Experimental Example 1, except that the microstructures of Experimental Example 1 were specified as concave-conical with a diameter of 300 pm, a farthest horizontal distance of 450 pm, a furthest horizontal distance close distance of 150 pm, a mean distance of 300 pm and a depth of 450 pm, and that the diameter of the latex particles was specified as 100 pm. In the fabricated membrane support, the convex portions were cone-shaped. The farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are shown in the Table.

[Experimental Example 12]

The experiment was carried out under the same conditions as in Experimental Example 1 except that, in Experimental Example 1, a 0.025% w/v suspension solution containing latex particles with a diameter of 0.5 pm and a 0.025% w/v suspension solution containing latex particles having a diameter of 1 pm in equal volumes, and a marker pad was prepared using the solution. In the fabricated membrane support, the convex portions were cone-shaped. The farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are shown in the Table.

[Experiment example 13]

The experiment was carried out under the same conditions as in Experimental Example 1 except that, in Experimental Example 1, a 0.025% w/v suspension solution containing latex particles with a diameter of 1 µm and a 0.025% w/v suspension solution containing latex particles having a diameter of 5 pm in equal volumes and a marker pad was prepared using the solution; and that the microstructures were specified as conical-concave with a diameter of 80 pm, a farthest horizontal distance of 100 pm, a nearest horizontal distance of 20 pm, a mean distance of 60 pm, and a depth of 100 pm. In the fabricated membrane support, the convex portions were cone-shaped. The farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are shown in the Table.

[Experimental Example 14]

The experiment was carried out under the same conditions as in Experimental Example 1 except that, in Experimental Example 1, a 0.025% w/v suspension solution containing latex particles with a diameter of 5 µm and a 0.025% w/v suspension solution containing latex particles having a diameter of 20 pm in equal volumes and a marker pad was prepared using the solution; and that the microstructures were specified as conical-concave with a diameter of 300 pm, a farthest horizontal distance of 450 pm, a nearest horizontal distance of 150 pm, a mean distance of 300 pm, and a depth of 450 pm. In the fabricated membrane support, the convex portions were cone-shaped. The farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are shown in the Table.

[Experimental Example 15]

The experiment was carried out under the same conditions as in Experimental Example 1 except that the diameter of the latex particles used in the microstructure of Experimental Example 1 was specified as 0.4 pm. In the fabricated membrane support, the convex portions were cone-shaped. The farthest horizontal distance, the nearest horizontal distance, the middle distance, and the diameter and height of the convex parts are shown in the Table.

[Experimental Examples 16 to 27]

The experiments were carried out under the same conditions as in Experimental Examples 3 to 14 except that the particles used were changed from colored latex particles to fluorescent latex particles (micromer-F fluorescent latex particles, material: polystyrene manufactured by Corefront Corporation); and that the dilution rate (maximum permissible dilution rate for fluorescence determination) at which an immunochromate reader (C11787 manufactured by Hamamatsu Photonics KK) cannot read the presence or absence of a colored line 10 minutes after the test start. Except for this, it was the same way as in Experimental Examples 3 to 14. In the fabricated membrane supports, the farthest horizontal distance, the nearest horizontal distance, the mean distance, the diameter of the fluorescent latex particles ( diameter of the fluorescent latex particle), and the diameter and height of the convex parts are shown in Table 2.

General evaluations were carried out based on the permissible dilution rates for fluorescent determination according to the following criteria and the results are shown in Table 2.

A: The allowable dilution rate for fluorescence determination is 5 x 104 or more.

B: The global evaluation of A and C does not apply.

C: The allowable dilution rate for fluorescence determination is 1 x 104 or more and 2 x 104 or less.

_

From the results shown in Tables 1 to 2, it was shown that the liquid sample test kit according to the embodiment enables highly sensitive detection by specifying the size of microstructures in the flow path and developing a marker. according to the size. If the particle diameter is small, the dilution limit becomes low and the sensitivity is low (Experimental Example 1, Experimental Example 15). If the particle diameter is large, a label cannot be developed and detection cannot be performed (Experimental Example 2).

Industrial Applicability

In the embodiment, in an immunochromatography, which allows optical confirmation that a target substance was detected, a test kit is provided that allows a highly sensitive determination. The liquid sample test kit according to the embodiment enables the implementation of a highly sensitive test at low cost and is therefore useful as a disposable POCT reagent. List of reference signs

2: Flow path, 3: Membrane support having microstructures provided therein, 3x: Placement zone, 3y: Detection zone, 4,4a,4b,4c,4d,4e: Representative length of bottom surface of a convex part (diameter of the bottom of the convex part), 5A: Farthest horizontal distance between adjacent microstructures (closest center-to-center distance), 5B: Closest horizontal distance between adjacent microstructures (distance between closest microstructures ), 5C: Distance of the space between adjacent microstructures, 6,6a,6b,6c,6d: Height of the convex parts, 7,7a,7b,7c,7d,7e: Microstructure, 8,8a,8b,8c,8d ,8e: Convex part, 9: Flat part, 10,10a,10b,10c,10d: Bottom surface of convex parts, 18: Test kit for liquid sample, 18a: Box, 18b: First opening, 18c: Second opening , d: Flow direction of the liquid sample (transport direction)

Claims (7)

1. A membrane support (3) for detecting a target substance in a liquid sample, the membrane support (3) comprising: a flow path (2), a microstructure (7) formed at the bottom of the flow path (2), a detection zone (3y) for detecting the target substance in the liquid sample; Y a marker provided in at least a part of the flow path (2) on an upstream side of the detection zone (3y), the marker being constituted by: a particle having a diameter of 500 nm or more and 100 pm or less, and an antibody or an antigen bound to the particle, wherein the antibody and the antigen are capable of reacting specifically with a target substance in the liquid sample, and a detection substance immobilized in the detection zone (3y) of the membrane support (3), such detection substance is capable of producing a color change by holding a target substance, such that the target substance to which it is is going to bind the marker can be detected in the detection zone (3y) and the color of the detection zone (3y) will change when the marker binds. 2. The membrane support (3) according to claim 1, wherein the average horizontal distance between the adjacent convex parts (8) that constitute the microstructure (7) is at least 3 times the diameter of the particle and 300 pm or less. The membrane support (3) according to claim 1 or 2, wherein the particle is one or more selected from the group consisting of a colored latex particle and a fluorescent latex particle. 4. The membrane support according to any of claims 1 to 3, wherein the detection zone (3y) is capable of producing a color change when the target substance is detected, in such a way that the target substance at the to be bound by the marker can be detected in the detection zone (3y) and where the color of the detection zone (3y) will change upon binding of the marker. 5. A method for manufacturing a membrane support (3) according to any of claims 1 to 4, comprising immobilizing the detection substance capable of producing a color change by maintaining a target substance in the detection zone (3y) of the membrane support (3) according to any of claims 1 to 4. 6. A test kit comprising a membrane support according to any one of claims 1 to 4. 7. A method for manufacturing a test kit according to claim 6.